Downregulation of muscarinic receptors gene expression in human breast cancer cellsregulates anchorage-independent cell growthin vitro and angiogenesis in vivo

Abstract

Downregulation of muscarinic receptors gene expression in human breast cancer cellsregulates anchorage-independent cell growthin vitro and angiogenesis in Muscarinic receptors (M) expression, activation and signalling play important roles in regulating many cellular process and cancer progression. It has been reported thathuman breast cancerMCF-7 cells express muscarinic receptors M3 and M4 subtypes and its activation promotes tumoral progression. We previously reported thatthe silencing of both M3 and M4in MCF-7 cells significantly reduced neovascularization capacity of tumoral cellsin vivo.The aim of this work was to evaluate the specific contribution of each M receptor on different tumoral progression parameters likeanchorage-independent cell growth and angiogenesis in vivo. Here, we silencedM3 or M4 subtypes in MCF-7 cells by specific RNAi. After 5 days we used the different experimental groups(siM3, siM4andMCF-7 cells with and without carbachol (Carb, -8M))in the following assays.Briefly, for soft agar colony assay we seeded 2X104 cells of each group into medium with soft agar. After 2 weeks, the colonies larger than 60µm in diameter were counted. We observed thatcholinergic stimulation of siMcells showed a significant reduction in colony number when compared withMCF-7+Carb, however this effect was greater in siM3 cells than in siM4 cells (siM3: 99.97±9.50%, siM4: 289.4±5.3% vsMCF-7: 509.1±11.8%;p<0.0001).Angiogenesis was measured by inoculation of 2x105 cells in female nude mice. After 5 days, the animals were sacrificed and angiogenesis was quantified in the sites of inoculation as vessel density.We found that silencing of both M receptors decreased the neovascular response in vivo of siM cells treated with Carb compared with MCF-7+Carb(siM3: 3.6±0.1, siM4: 3.7±0.3 vs MCF-7: 6.4±0.7; p<0.0001).According to our results,Mreceptors expressiondownregulation can modulate the malignant phenotype of MCF-7 cells, having a high inhibitory effect on anchorage-independent cell growth andangiogenesis.